TECHNICAL NOTE Examining root nodule activity on legumes In brief The symbiotic partnership between bacteria and leg‐
umes is vital for the supply of biologically fixed nitro‐
gen to soil and plants in organic cropping systems. Perennial clovers in a dense grass clover ley may contribute more than 200 kg nitrogen (N) per ha and year to the cropping system from the second year after sowing. This technical note aims at encouraging farmers to take a closer look at the roots of legumes and be‐
come more familiar with the process of nitrogen fix‐
ation and its relevance for organic farming. The note provides some key information on biological nitro‐
gen fixation and describes a simple method for exa‐
mining nitrogen‐fixing root nodules. Biological nitrogen fixation Symbiosis of plant roots and bacteria In most European soils, clover, alfalfa, beans, peas and vetch form a partnership (symbiosis) with native Rhizo‐
bium bacteria strains. Soybean and lupine form a sym‐
biosis with Bradyrhizobium bacteria, which are often missing in Europe soils. If the bacteria strains are not native in the actual soil, inoculation with the right bac‐
teria (on seeds) is required prior to sowing. An intricate communication between the plant roots and the free‐living stage of the bacterium initiates a nodulation on the roots, in which the bacteria multiply. The bacteria in the nodules on legume roots biologically convert nitrogen from inert atmospheric N2 gas to am‐
monia and amino acids within the nodules. The process requires energy, which the bacteria receive from the plant. Thus, factors hampering the photosynthesis in plants, as water stress and lack of other nutrients than nitrogen, will also reduce the biological fixation of ni‐
trogen. As a total, the symbiosis between the plant in good growth and the bacteria enhances the nitrogen content in the whole plant‐soil system. Nodule size and shape can differ from one plant species to another (1). Large, round nodules are usually found on soybean (Glycine max) and faba bean (Vicia faba). Smaller, more elongated nodules are present on clover species (Trifolium spp.) and alfalfa (Medicago). Some plant species have more asymmetrically shaped nod‐
ules. Active nodules are red coloured inside Actively N‐fixing nodules contain a pigmented protein called leghaemoglobin. Its presence results in a red col‐
ouration of the interior of nodules, indicating that the bacteria are alive and active (2). Dead, inactive and se‐
nescent nodules are usually greyish green or brown in‐
side. Maximum nitrogen fixation around flowering Nodules appear 4 to 6 weeks after sowing and reach maximum activity around flowering. In autumn and at plant maturity (after flowering), the roots and nodules are senescent and some have started to decay. When the perennial crop, e.g. clover ley, is cut, nitrogen fixa‐
tion may continue throughout the season. In perennial legumes, new nodulation starts again in spring. This publication was produced in the framework of the FertilCrop project funded by CORE Organic Plus funding bodies being partners of the FP7 ERA‐Net project CORE Organic Plus. 1
Grasses grown in mixture with forage legumes can use up to 80 % of the nitrogen from the N fixation in the legumes. In grazed white clover pasture, a fixation rate of 55 to 295 kg N per ha and year was recorded above ground (4). Active, red coloured nodule on lupin (Lupinus). (Photo: R. Pom‐
meresche, NORSØK) Nodules on soybeans with active (red colour) bacteria (Bradyrhizo‐
bium) (Photo: Monika Messmer, FiBL). Older, more senescent and inactive nodules with grey colour, on Hungarian vetch (Vicea pannonica). (Photo: R. Pommeresche, NORSØK) In soybeans under irrigation, average N fixation is about 175 kg N per ha and year, measured in aboveground plant‐
tissue nitrogen (250 kg N if roots were included), and about 100 kg N without irrigation (5). Lupines fix about 165 kg N in aboveground tissue, and faba beans about 90 kg per ha (5) (about 130 kg per ha and year, if roots were included). As nitrogen fixation is sensitive to water stress, it will be lower during summertime in dry areas without irrigation. The optimal temperature for several Rhizobium bacteria strains is around 20 °C, but for many legumes and regions, fixations at much lower temperatures are recorded. Nitro‐
gen fixation per ha and year in northern areas (around 60° N) and areas with a milder climate (around 40° N) (3) was noticed to be almost the same. The reason may be that sev‐
eral environmental factors are involved in these processes. Brown or “empty” nodules from red clover (Trifolium pratense). (Photo: R. Pommeresche, NORSØK). Wide range of N fixation rates Nitrogen fixation in a legume‐rhizobia symbiosis varies widely according to climate, soil nitrogen status, cropping system and management. The amount of fixed nitrogen in above‐ground plant tissues of ungrazed forage legumes in temperate/boreal areas ranges from 60 to 370 kg N per ha and year in red clover (Trifolium pratense L.), from 80 to 540 kg N per ha and year in white clover (T. repens L.) and from 100 to 350 kg N per ha and year in alfalfa (Medicago sativa L.) (3). Large proportion of nitrogen in root biomass A high proportion of fixed nitrogen is found in the roots and nodules of the legume plants. Pot experiments indicate that 20 to 50 % of the total N in alfalfa (Medicago spp.), faba beans (Vicia spp.) and lupines (Lupinus spp.) is present in roots (in‐
cluding nodules) at the time of flowering. However, the allo‐
cation of the nitrogen can vary widely between shoots and roots within the same genus (Medicago spp., Lupinus spp.) (5). ln a red clover‐grass ley, 60 % of the nitrogen was found in the clover stubble and 25 % in the roots after the first and second harvest, while about 60 % of the N in the grasses re‐
mained in stubble and roots after second harvest (6). Pommeresche & Hansen (2017): Examining root nodule activity on legumes. FertilCrop Technical Note. Download at www.fertilcrop.net 2
5. Look for nodules on the roots and examine, whether they are active or not by cutting three nodules of each plant in half immediately after uprooting. 6. Repeat the procedure with another plant of the same spe‐
cies, but choose another part of the field, which is either better or worse in growth. Useful questions  Appearance of the plant: Does it look lush with dark green coloured leaves, or does it rather look meagre with fewer and pale green leaves?  Development of the roots: How well did the root system develop? Are the roots long and frequently branched? Red and with clover roots with the shape and colour of nitrogen fixing nodules. Active, pink nodules on red clover roots (upper) and a close up of the interior red colour of a nodule on with clover. Both from meadows in Norway. (Photos: R. Pommeresche, NORSØK). Different number and distribution of nodules on roots of dif‐
ferent plant species The location of the nodules along the roots is primarily linked to genus/species of the plants and the presence of rhizobia in the soil. At low frequency of rhizobia, the low number of nod‐
ules per plant is compensated by increased nodule size. In ad‐
dition, the content of nutrients in the soil, local soil structure and access to air in the soil influence the distribution and size of the nodules. Therefore, comparisons of nodules should be done within the same plant species and plant stage.  Number, size and distribution of root nodules: Can you find nodules on the roots? Are there only few nodules or are there many? Are the nodules of regular size or very large? Have the nodules developed on the main root (tap‐
root) only or also on lateral roots? Ideally, nodules are found on different roots. Are the nodules only on the up‐
per third or also on the lower two thirds of the root sys‐
tem? Ideally, nodules are found on the whole root depth.  Colour of root nodules: Which colour do the nodules have that have been cut in half? Red/purple/pinkish colour = active bacteria; greyish green or brown colour = old or in‐
active bacteria; white = young nodules or nodules in pro‐
gress to become active or inactive. Investigation method The following method describes a simple procedure to be‐
come familiar with legume‐rhizobia symbiosis and to get a first feedback on the activity of the symbionts in the field. How to proceed 1. Optimally, two weeks before the legumes are flowering (or any time in the growing season) select two to four leg‐
ume plants from a representative part of a field. 2. Dig out a soil cube (about 25 x 25 cm and as deep as the roots or the spade go) around a chosen legume plant. 3. Lift the clump out carefully with the spade and put it on a plastic sheet. 4. Carefully remove the soil from the roots manually and try to loosen the legume roots with a fork. On heavy soil, wa‐
ter might be needed to remove the soil without destroy‐
ing nodules. Searching for root nodules in a clover ley, Norway. We found nod‐
ules and active nitrogen‐fixating bacteria on some of the red clover roots (Photo: S. Hansen, NORSØK). Required material  A spade (to dig out the soil cubes with plant roots)  Some plastic sheets (to put the soil and plants on)  A pen, some paper, a fork, a ruler and a camera  A bucket of water (to remove soil from plant roots) Pommeresche & Hansen (2017): Examining root nodule activity on legumes. FertilCrop Technical Note. Download at www.fertilcrop.net 3
Interpretation of the findings Observations Possible conclusions and recommendations Colour and number of  Regardless of the plant species, if about 15 nodules are found on the roots of one plant and most
nodules of them are red inside, it can be assumed that the nitrogen‐fixating bacteria are performing well.  Generally, Rhizobia and Bradyrhizobia bacteria are found in the soil if the plant genus is found nat‐
urally in the area, or if the legume genus has successfully been grown on the field before. If the
matching bacteria are missing in the soil, most legumes will show suboptimal growth. In such cases,
inoculation with the matching bacteria is indispensable for optimal plant performance. Lack of the
bacteria cannot be compensated by N fertilisation. N‐fertilization affects nodulation negatively.  After successful inoculation and repeated cultivation of the same legume species, the number of
nodules can exceed 100 per plant. However, the number of nodules is not equivalent to a high
fixation rate, but a precondition for efficient symbiosis. Size of the nodules  Evenly distributed nodules of regular size often indicate higher nitrogen fixation rates than the pres‐
ence of a few, very large nodules. Distribution of the nodules on the roots  Nodules in the upper root zone only, may indicate shortage of air in the lower root zone. In such
cases, better soil structure may improve aeration of the lower root zone and access to nitrogen and,
as a result of it, may induce development of nodules in deeper soil layers. Nodules only on the tap/main root  Nodules on the tap or main root only, may indicate that the bacteria try to compensate low fixation
by increasing their number inside the nodules. 3 Carlsson, G. & Huss‐Danell, K. 2003. Nitrogen fixation in peren‐
nial forage legumes in the field. Plant and Soil 253, 353‐372. 4 Ledgard, S.F. & Steele, K.W. 1992. Biological nitrogen fixation in mixed Iegume/grass pasture. Plant and Soil 141,137‐153. 5 Unkovich, M.J. & Pate, J.S., 2000. An appraisal of recent meas‐
urements of symbiotic N2 fixation by annual legumes. Field Crops Research 65, 211‐228. 6 Huss‐Danell, K., Chaia, E. & Carlsson, G., 2007. N 2 fixation and nitrogen allocation to above and below ground plant parts in red clover grasslands. Plant Soil 299, 215‐226. References 1 Somasegaran, P. & H.J. Hoben. 1994. Handbook of Rhizobia. Methods in Legume‐Rhizobium Technology. Springer Verlag, New York, lnc. 2 Virtanen, A. l., Jorma, J., Linkola, H. & Linnasalmi, A. 1947. On the relation between nitrogen fixation and leghaemoglobin con‐
tent of leguminous root nodules. Acta Chem. Scan. 190‐111. Pdf. Imprint Publishers Research Institute of Organic Agriculture FiBL Ackerstrasse 113, Postfach 219, CH‐5070 Frick, Switzerland Phone +41 62 865 72 72, [email protected], www.fibl.org Norwegian Centre for Organic Agriculture (NORSØK) Gunnars veg 6 , NO‐6630 Tingvoll, Norway Phone +47 930 09 884, [email protected], www.norsok.no Authors Reidun Pommeresche and Sissel Hansen (both NORSØK). Review Andreas Fliessbach, Christine Arncken‐Karutz, Monika Messmer, Kathrin Huber, Gilles Weidmann (all FiBL) Language editing Andreas Basler (FiBL) Photo cover page Soybean roots with nitrogen fixing bacteria in nodules. Photo: Monika Messmer (FiBL) Download This technical note is available at www.fertilcrop.net. © Research Institute of Organic Agriculture, Switzerland, 2017 About FertilCrop Fertility Building Management Measures in Organic Cropping Systems – FertilCrop is a project funded by the CORE Organic Plus funding bodies, being partners of the FP7 ERA‐Net project CORE Organic Plus. The overall aim of FertilCrop is to develop efficient and sustainable management techniques aimed at in‐
creasing crop productivity in organic farming systems. More in‐
formation about FertilCrop is available at www.fertilcrop.net. Disclaimer The contents of this technical note are the sole responsibility of the authors, and they do not represent necessarily the views of the project funders. Whilst all reasonable effort is made to en‐
sure the accuracy of information contained in this technical note, it is provided without warranty and we accept no respon‐
sibility for any use that may be made of the information. Pommeresche & Hansen (2017): Examining root nodule activity on legumes. FertilCrop Technical Note. Download at www.fertilcrop.net 4